types of genetic markers multilocus markers (rapd, aflp, minisatelitte dna fingerprinting)...
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Types of genetic markers
• multilocus markers (RAPD, AFLP, minisatelitte DNA fingerprinting)
• single-locus markers (allozymes, microsatelittes, SNPs)
• dominant markers – scored as present or absent (RAPD, AFLP, ...)
• codominant markers – identification of homologous alleles, i.e. scoring of homozygote and heterozygote states (allow estimation of allele frequencies – SNPs, microsatelittes, ...)
Main markers used in molecular ecologySingle locus
Codominant PCR assay Overall variability
Mitochondrial DNA (or sex specific sequences like Y chromosome)
Sequences Yes Haplotypes Yes Low-high
Nuclear multilocus
Minisatellite fingerprints
No No No High
RAPD No No Yes High
AFLP No No Yes High
Nuclear single locus
Allozymes Yes Yes No Low-medium
Microsatellites Yes Yes Yes High
SNPs (sequences) Yes Yes Yes Low-high
Multi-locus genetic markers• screening of many loci distributed
randomly throughout the genome
minisatellite DNA fingerprinting
RAPD (randomly amplified polymorphic DNA)
AFLP (arbitrary or amplified fragment length polymorphism)
• presence vs. absence - codominant
Př.: chromozóm 1
Minisatellite DNA fingerprinting
• randomly distributed repetitions (Alu sekvence, SINE, LINE)
• restriction – sequence specific endonucleases
• electrophoresis
• blotting
• hybridization with probe
• over recent years – shift to PCR-based methods
RAPD (randomly amplified polymorphic DNA)
short random oligonucleotides as primers
PCR at low stringency
detection of PCR fragments by electrophoresis
-
+
low repeatability due to many factors affecting PCR – is not more accepted as method for studies of population structure
AFLP (amplified fragments length polymorphism)
• cheap, easy, fast and reliable method to generate hunderds of informative genetic markers
• simultaneous screening of many different DNA regions distributed randomly throughout the genome
• more reproducible banding pattern than RAPD
Generating AFLP markers
Generating AFLP markers
multi-locus genotype
„capillary version“
PCR with primers on adaptors
Single-locus genetic markers
• allozymes and other transcribed genes
• SNPs (single nucleotide polymorphisms)
• microsatelittes (length polymorphism)
Př.: chromozóm 1
Single nucleotide polymorphisms (SNPs)
SNPs : nuclear genome (consensus)
Example of SNP marker
transiceA ↔ G
transition: PuPu or PyPy transversion: PuPy or PyPu
Use of SNPs markers
• species (or genetical group) identification and analysis of hybridization
• phylogeography• population genetics (genetic variation,
individual identification – parentage, relatedness, population structure, population size, changes in population size)
Advantages
• abundant and widespread in many genomes (in both coding and non-coding regions) – milions of loci
• spaced every 300-1000 bp
• biparentaly inherited (vs. mtDNA)
• evolution is well described by simple mutation models (vs. microsatellites)
• shorter fragments are needed – using in non-invasive methods
Disadvantages
• ascertainment bias – selection of loci from an unrepresentative sample of individuals
• low variability per locus (usually bi-allelic)
• higher number of loci is needed in population genetic applications (4-10 times more loci)
Methods
1. Locus discovery (ascertainment)
2. Genotyping
SNPs discovery
CATS loci = comparative anchor tagged site loci (= cross amplification)
Genomic library = genome restriction + cloning
AFLP = alternative to the genomic library construction (provide PCR fragments, can be transformed to informative SNP)
Sequencing
DNA
PCR product
cloned fragment
sequencing reaction with marked
dideoxynucleotides and specific or universal
primers
GA
GT
GC
C
-+
laser beam
detector
capillary electrophoresis
Sangrova dideoxy metoda
• Sekvence délky 500 – 1000 bp
• 4 kapiláry - destička s 96 vzorky za noc
• Jsou i sekvenátory s 96 kapilárami
Použití nových přístupů
Metagenomické knihovny Ursus spelaeus> 28 000 bp (jaderná i mitochondriální DNA)(Noonan et al. 2005)
sekvenování
„Next generation“ sequencing (Hudson 2008)
„polonies“(polymerase colonies)
454 pyrosequencing
• emulzní techniky amplifikace pikolitrové objemy
• simultánní sekvenování na destičce z optických vlákendetekce pyrofosfátů uvolňovaných při inkorporaci bazí
• První generace GS20 → 200 000 reakcí najednou (zhruba 20 milionů bp) dnes FLX → 400 000 reakcí najednou
• Problémy s homopolymery
• Délka jednotlivých sekvencí 100 – 400
1 600 000 well plate
Solexa/Illumina 1G SBS technology(SBS = sequencing by synthesis)
• 1 Gb (šestinásobek genomu Drosophily)
• Výrazně levnější
• Sekvence délky 35 bp
• Flourescence, reversibilní terminátory
• Spíš pro resequencing
SOLiD(sequencing by Oligonucleotide Ligation and Detection)
SNP genotyping - old standardsPCR-RFLP
(restriction fragments length polymorphism)
CCGATCAATGCGGCAAGGCTAGTTACGCCGTT
CCGATCACTGCGGCAAGGCTAGTGACGCCGTT
cutting by restriction endonuclease
Allele 1
Allele 2
-
+allele 1 allele 2
PCR-SSCP (gel or capillary)(single strand conformation
polymorphism)
Allele 1 Allele 2
FAM
HEX
allele 1
allele 2
+ -
no cut
SNP genotyping – new methods1) ASPE: allele-specific primer extension
CCGATCAATGCGGCAA
CCGATCAATGCGGCAA
T
G
• primer extension with deoxy nucleotides and highly specific polymerase enables allele-specific amplification
• 3’ terminal nucleotide of the two primers contains the SNP nucleotide
• two PCRs with specific primers are necessary
succesfull PCR
no PCR product
SNP genotyping – new methods
2) SBE: single base extension
CCGATCAATGCGGCAA
CCGATCACTGCGGCAA
T
G
only one dideoxy nucleotide is added to the primer
T
G
+ -
Detection or SBE products
+ -
electrophoresis in a capillary
microarrays – multicolor detection(using of 5’ oligonucleotide tags on SBE primers)
CCGATCACTGCGGCAA
Gtagother methods: flow cytometry, fluorescence polarization
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